NYD-SP15: a novel gene potentially involved in regulating testicular development and spermatogenesis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, we identified a novel human testis gene, NYD-SP15. NYD-SP15 expression was 3.26-fold higher in adult than in fetal testis; however, there was almost no NYD-SP15 expression in the sperm. NYD-SP15 comprises 3364 base pairs, including a 1545 bp open reading frame encoding a 514 amino acid protein possessing 89% sequence identity with the mouse testis homologous protein. NYD-SP15 is located on human chromosome 13q14.2. The deduced structure of the protein contains two dCMP_cyt_deam domains, indicating a potential functional role for zinc ion binding. The gene is expressed variably in a wide range of tissues, with high expression levels in the testis. Sequence analysis revealed that NYD-SP15 is not a highly conserved protein, with its distribution in high-level species such as vertebrates including Homo, Mus, Rattus, and Canis. The results of semiquantitative polymerase chain reaction in mouse testis representing different developmental stages indicate that NYD-SP15 expression was developmentally regulated. These results suggest the putative NYD-SP15 protein may play an important role in testicular development and spermatogenesis and may be an important factor governing male infertility.     

Molecular cloning, identification and characteristics of a novel isoform of carbamyl phosphate synthetase I in human testis

A gene coding a novel isoform of carbamyl phosphate synthetase I (CPS1) was cloned from a human testicular library. As shown by cDNA microarray hybridization, this gene was expressed at a higher level in human adult testes than in fetal testes. The full length of its cDNA was 3831 bp, with a 3149 bp open reading frame, encoding a 1050-amino-acid protein. The cDNA sequence was deposited in the GenBank (AY317138). Sequence analysis showed that it was homologous to the human CPS1 gene. The putative protein contained functional domains composing the intact large subunit of carbamoyl phosphate synthetase, thus indicated it has the capability of arginine biosynthesis. A multiple tissue expression profile showed high expression of this gene in human testis, suggesting the novel alternative splicing form of CPS1 may be correlated with human spermatogenesis.     

NYD-SP16, a novel gene associated with spermatogenesis of human testis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, a novel human testis gene NYD-SP16 was identified. NYD-SP16 expression was 6.44-fold higher in adult testis than in fetal testis. NYD-SP16 contains 1595 base pairs (bp) and a 762-bp open reading frame encoding a 254-amino acid protein with 73% amino acid sequence identity with the mouse testis homologous protein. The NYD-SP16 gene was localized to human chromosome 5q14. The deduced structure of the NYD-SP16 protein contains one transmembrane domain, which was confirmed by GFP/NYD-SP16 fusion protein expression in the cytomembrane of the transfected human choriocarcinoma JAR cells, suggesting that it is a transmembrane protein. Multiple tissue distribution indicated that NYD-SP16 mRNA is highly expressed in the testes and pancreas, with little or no expression elsewhere. Further analysis of abnormal expression in infertile male patients revealed complete absence of NYD-SP16 in the testes of patients with Sertoli-cell-only syndrome and variable expression in patients with spermatogenic arrest. Homologous gene expression in mouse testis was confirmed in spermatogenic cells by in situ hybridization. The results of cDNA microarray, in situ hybridization, and semiquantitative polymerase chain reaction in mouse testis of different stages indicated that NYD-SP16 expression is developmentally regulated. These results suggest that the putative NYD-SP16 protein may play an important role in testicular development/spermatogenesis and may be an important factor in male infertility.     

Cloning and identification of a novel RNF6 transcriptional splice variant Spg2 in human development

RING finger proteins are zinc finger proteins containing the RING motifs. They act mainly as E3 ubiq-uitin ligases, bind the ubiquitin E2 conjugating enzyme and promote degradation of targeted proteins, Many novel genes have been isolated and differentially expressed in human adult and embryo testis by a testis cDNA-array differential display technique. A novel RING finger cDNA is highly expressed in adult testis and at low level in fetal testis. It was named Spg2. It contains a 2055 nucleotide ORF, en-codes a 685-amino-acid RNF6 protein, and has a RING finger in its C terminal. NCBI Blast shows that the gene is located on chromosome 13 and contains five exons. A multiple tissue expression profile also indicates that it is highly expressed in human testis, so we speculate that it may be associated with human spermatogenesis by virtue of the action of its RING domain.     

NYD-SP15: A Novel Gene Potentially Involved in Regulating Testicular Development and Spermatogenesis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, we identified a novel human testis gene, NYD-SP15. NYD-SP15 expression was 3.26-fold higher in adult than in fetal testis; however, there was almost no NYD-SP15 expression in the sperm. NYD-SP15 comprises 3364 base pairs, including a 1545 bp open reading frame encoding a 514 amino acid protein possessing 89% sequence identity with the mouse testis homologous protein. NYD-SP15 is located on human chromosome 13q14.2. The deduced structure of the protein contains two dCMP_cyt_deam domains, indicating a potential functional role for zinc ion binding. The gene is expressed variably in a wide range of tissues, with high expression levels in the testis. Sequence analysis revealed that NYD-SP15 is not a highly conserved protein, with its distribution in high-level species such as vertebrates including Homo, Mus, Rattus, and Canis. The results of semiquantitative polymerase chain reaction in mouse testis representing different developmental stages indicate that NYD-SP15 expression was developmentally regulated. These results suggest the putative NYD-SP15 protein may play an important role in testicular development and spermatogenesis and may be an important factor governing male infertility. These authors contributed equally to this work     

Identification and characteristics of a novel E1 like gene nUBE1L in human testis

A gene, presumably involved in spermatogenesis, was identified and characterized by using cDNA microarray. Hybridization intensity was 2.13 fold higher in adult testis than that in fetal testis.The full length of this gene was 4288bp and it encoded a 578 amino acid protein. Conserved structure and amino acid sequence analysis revealed that the protein contained 1 Thif-domain, 2 UBACT-domains,and a functional active site cysteine lay upstream of UBACT domain, all of them also existed in ubiquitin-activating enzyme E1 and E1 like proteins. So we named this gene as a novel ubiquitin-activating enzyme E1 like gene (nUBE1L). Expression profile showed that nUBE1L was predominantly expressed in testis.Comparison of the expression of nUBE1L in different developmental stages of testis indicated that it was highly expressed in adult testis. In conclusion, nUBE1L is a novel human E1 like gene highly expressed inadult testis, which plays key role in ubiquitin system, and accordingly influences spermatogenesis and male fertility.     

睾丸生精细胞凋亡相关基因TSARG1与Mtsarg1的分子克隆及Mtsarg1基因的表达谱分析

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段（GenBank登录号：BE644538）出发,利用生物信息学和实验技术,克隆了小鼠睾丸生精细胞凋亡相关新基因Mtsarg1及相应的人类新基因TSARG1,Gen-Bank登录号分别为AF399971和AY032925。小鼠Mtsargl与人类TSARGl基因在氨基酸水平有55％的一致性和61％相似性,与其他已知蛋白质无明显同源性。小鼠10种组织的RT-PCR分析结果表明,Mtsargl基因在睾丸中高表达,在附睾中呈微弱表达,在其他组织不表达,提示Mtsargl和TSARGl基因在生精细胞凋亡或精子发生中具有潜在的重要作用。     

In‐depth proteomic analysis of whole testis tissue from the adult rhesus macaque

The rhesus macaque is similar to humans both anatomically and physiologically as a primate, and has therefore been used extensively in medical and biological research, including reproductive physiology. Despite sequencing of the macaque genome, limited postgenomic studies have been performed to date. In studies aimed at characterizing spermatogenesis, we successfully identified 9078 macaque testis proteins corresponding to 8662 genes, using advanced MS and an optimized proteomics platform, indicative of complex protein compositions during macaque spermatogenesis. Immunohistochemistry analysis further revealed the presence of proteins from different types of testicular cells, including Sertoli cells, Leydig cells, and various stages of germ cells. Our data provide expression evidence at protein level of 3010 protein‐coding genes in 8662 identified testis genes for the first time. We further identified 421 homologous genes from the proteome already known to be essential for male infertility in mouse. Comparative analysis of the proteome showed high similarity with the published human testis proteome, implying that macaque and human may use similar proteins to regulate spermatogenesis. Our in‐depth analysis of macaque spermatogenesis provides a rich resource for further studies, and supports the utility of macaque as a suitable model for the study of human reproduction.     

Normal sexual development and fertility in testatin knockout mice

The testatin gene was previously isolated in a screen focused on finding novel signaling molecules involved in sex determination and differentiation. testatin is specifically upregulated in pre-Sertoli cells in early fetal development, immediately after the onset of Sry expression, and was therefore considered a strong candidate for involvement in early testis development. testatin expression is maintained in the adult Sertoli cell, and it can also be found in a small population of germ cells. Testatin shows homology to family 2 cystatins, a group of broadly expressed small secretory proteins that are inhibitors of cysteine proteases in vitro but whose in vivo functions are unclear. testatin belongs to a novel subfamily among the cystatins, comprising genes that all show expression patterns that are strikingly restricted to reproductive tissue. To investigate a possible role of testatin in testis development and male reproduction, we have generated a mouse with targeted disruption of the testatin gene. We found no abnormalities in the testatin knockout mice with regard to fetal and adult testis morphology, cellular ultrastructure, body and testis weight, number of offspring, spermatogenesis, or hormonal parameters (testosterone, luteinizing hormone, and follicle-stimulating hormone).     

Electroporated transgene-rescued spermatogenesis in infertile mutant mice with a sertoli cell defect

The molecular basis of most human male infertility arising from spermatogenesis disruption is poorly understood because of a lack of useful investigation systems. To study the roles of the supporting Sertoli cells in mammalian spermatogenesis, we improved an electroporation technique for seminiferous tubules in vivo. Because Sertoli cells barely proliferate in mature testis, linear transgenes are not incorporated into the genome and quickly degrade. However, circular expression vector is stably expressed in Sertoli cells for a long period. By electrotransformation of a complete cDNA, we rescued defective spermatogenesis in infertile Sl(17H)/Sl(17H) mutant mice with partial dysfunction of stem cell factor in Sertoli cells. Application of this gene transfer system will facilitate both the understanding of spermatogenesis and the development of new gene therapies for human male infertility.     

人类生精相关基因TSARG4的cDNA克隆

为了探索精子生成的分子机制 ,从人精子外部致密纤维蛋白相关基因SPAG4(spermantigen 4)和小鼠精母细胞中表达的AK0 0 62 2 5基因出发 ,找到两个人类EST ,BG72 0 5 64和AI70 0 45 4,其中BG72 0 5 64在人睾丸中表达。运用“间隙填充法”填平这两个EST之间的间隙 ,从人睾丸文库中快速克隆了同源于SPAG4和AK0 0 62 2 5基因的人类TSARG4基因 (testisandspermatogenesisrelatedgene 4) (GenBank登录号为AF40 13 5 0 ) ,并用RT PCR对该基因阅读框进行验证。TSARG4基因全长 12 5 2bp ,开放阅读框为 94～ 12 3 3bp ,定位于 2 0q11.2 ,推定编码 3 79个氨基酸 ,预计分子量为 43 0 81.45 ,等电点为 8.61,该基因与小鼠精母细胞基因AK0 0 62 2 5编码的氨基酸序列同源性 74% ,与人类SPAG4基因编码的氨基酸序列同源性 45 %。RT PCR表明人类TSARG4基因在多个组织中均有表达 ,而同源的小鼠AK0 0 62 2 5基因仅在睾丸中表达